Proteus species are second only to Escherichia coli as the most common causative agent of Gram-negative based urinary tract infections and many harbor several virulence factors that provide inherent uropathogenicity. One of the virulence factors is a hemolysin system comprised of hemolysin A (HpmA) and hemolysin B (HpmB). The hemolysin (A/B) system within Proteus mirabilis belongs to a two-partner secretion (TPS) pathway found in various Gram-negative bacteria. TPS pathways are responsible for the secretion of large exoproteins, including the Ca2+-independent cytolysins/hemolyins from Serratia marcescens, Edwardsiella tarda, Yersinia pestis and Proteus mirabilis. HpmA is secreted and activated by HpmB allowing Proteus to lyse red blood cells. The overarching goal of the research aims to determine the mode of HpmA activation. A purified N-terminal fragment of HpmA (trcHpmA) was found sufficient to activate full-length inactive hemolysin A (HpmA*) and restore in vitro hemolysis. Both a CxxC and a deacetylase motif have been identified within the N-terminal fragment. The specific aims seek to examine the function of the CxxC, deacetylase and amidation motifs in the activation of HpmA. First, a CxxC motif was located within the amino terminal domain between cysteine 144 and cysteine 147. CxxC motifs have been shown to be involved within metal binding and disulfide isomerase sites. The research objectives will characterize the CxxC motif for both metal binding capacity and disulfide isomerase activity. In addition, two GKK amidation sequences were predicted between positions 1190 - 1193 and 1281 - 1284 of the C-terminal domain. Amidation sites have been shown to be instrumental during the activation of bioactive peptides. An additional part of the research objectives aim to analyze for proteolytic processing of HpmA via an amidation type mechanism during the activation of hemolysis. Collectively, the aims may determine if the CxxC, deacetylase, and amidation motifs have functional roles during the activation of HpmA. The results may provide information regarding the hemolysin virulence factor within Proteus mirabilis and allow new drug design strategies for the treatment of Proteus-based urinary tract infections.